The immune system provides defence for the body against infectious agents such as bacteria, viruses, fungi and parasites. More recently, it has been shown that the state of the immune system can be linked to the susceptibility to developing certain types of cancers. For example, it has been shown that in certain immune deficient states, most notably Acquired Immune Deficiency Syndrome (AIDS), the incidence of cancer is greatly increased. Immunotherapy has become an important therapy against various infectious agents and even in the treatment of cancer. One branch of immunotherapy involves using immunomodulating agents that can modulate, induce or increase the immune response.
One group of immunomodulating agents that has been extensively studied includes the cytokines. Cytokines are the chemical messengers of the immune system and are produced by immune cells such as lymphocytes and monocytes. One cytokine, namely interferon (IFN), has been shown not only to induce the immune system but also to act directly on tumors to inhibit proliferation of the tumor cells. Interferon is well known in the art. Details on its characterization as well as methods for its preparation can be found in Methods in Enzymology, Volume 119, "Interferons, PART C" 1986.
Although both crude as well as purified preparations of cytokines, such as interferon, are currently available, the problems with these known preparations include the following. Crude preparations generally contain proteinases which break down the cytokines and thereby decrease the stability of the preparation. Proteinase inhibitors are available but these are generally toxic or antigenic to man and thus are not useful in therapeutic applications. On the other hand, while purified interferon is generally proteinase free, the methods for purifying interferon known to date generally result in a poor yield of interferon. Furthermore, the biological activity of a purified preparation is generally lower than that of a crude interferon preparation.
Early in the 80s, it was observed that a crude preparation of interferon activated human natural killer cells (NK) more effectively than purified IFN prepared from the same batch of crude IFN. As shown in Table A the highest degree of NK activation can be achieved with a crude IFN titer of 50 IU/ml and a purified IFN titer of 625 IU/ml. These titers resulted in activation of the NK cells of 550% and 600%, respectively. From this data it was concluded that crude IFN has a higher potential of immunoactivation which is likely due to the presence of some other cytokines together with IFN.
It was later found that mixed cultures of donor Leukocytes produced (in response to viral induction and interaction of cells with different genotypes) cytokines as such as Interleukin-1 (IL-1), Tumor Necrosis Factor (TNF), Migration Inhibition Factor (MIF), Leucocyte Migration Inhibition Factor (LIF), together with IFN-.alpha.. It is known that these cytokines participate in the activation of immune effector cells (mainly macrophages, T.sub.4 -lymphocytes, neutrophils) and participate in the immune recognition of antigen and its elimination from the body. These reactions present the first stage of the immune response manifested by effectors in response to an antigen encounter. Amplification of immune response, developing specific humoral reactions and memory cells are events that occur later and depend on effective immune reactions in the first (non-specific) stage of immune response.
The cytokines such as IFN, IL-1, TNF, MIF and LIF, have different physico-chemical properties, therefore it is very difficult to retain composition that is active and composed of a mixture of the cytokines preparation after chemical purification procedures. Furthermore, for medical preparations containing protein it is very important to limit the quantity of total protein because foreign proteins (especially when used by parenteral routes) can induce sensitization. However, lowering of the level of protein increases the sensitivity of the active factors to proteolysis. As a result, the activity of such preparations after technological procedures is-decreased and problems with storage of the drug forms appear.
Therefore, in view of all of the above it is desirable to produce a cytokine composition that includes a mixture of cytokines and that is largely substantially free of proteinases while at the same time showing good biological activity.